Project Summary Alzheimer?s disease (AD) is the most common neurodegenerative disorder and a leading cause of disability and death. However, the molecular mechanisms underlying AD pathogenesis remains to be elucidated. Among the three human apolipoprotein (Apo) isoforms, ApoE4 is associated with increased risk for AD, ApoE3 is neutral, while ApoE2 is protective. Although there is a strong association of ApoE2 with reduced risk for AD, it remains unclear how ApoE2 modulates the AD pathological phenotypes and what are the molecular pathways mediating the neuroprotective effects of ApoE2. Filling this knowledge gap could help us to harness the power of ApoE2 to develop novel therapeutic strategies for effective treatment of AD and promoting healthy aging. Since the advent of induced pluripotent stem cell (iPSC) technology, human iPSCs (hiPSCs) have been widely used for disease modeling. Multiple studies have reported modeling AD using human iPSCs. Because hiPSCs are considered phenotypically young, hiPSCs have been used to capture early events in AD pathogenesis. Direct reprogramming is another type of reprogramming that allows direct conversion of one type of somatic cells into another. The direct reprogramming approach enables generation of human neurons that possess key elements of cellular aging, because this process does not go through the iPSC stage that involves extensive epigenetic modifications. Therefore, directly reprogrammed cells could provide a cellular platform that allows us to model late events of age-related, late-onset diseases, such as late-onset AD. The objective of this proposal is to define the role of ApoE2 in cellular functions associated with AD pathologies, and uncover molecular pathways that mediate the effects of ApoE2 in reducing the risk for AD, using human cellular models created through hiPSCs or direct reprogramming in combination with CRISPR/Cas9-mediated gene editing. We propose to establish cellular models for AD using both neurons and astrocytes derived from hiPSCs or direct reprogramming. We will determine how ApoE2 modulates AD pathological phenotypes in neuron-astrocyte co-cultures. Moreover, we will determine the relationship of ApoE2 genotype with gene expression levels in human brains and validate the findings obtained from our cell culture studies in human brain tissues. We hypothesize that ApoE2 protects neural cells from developing AD pathological phenotypes to reduce the risk for AD. Accordingly, we propose the following Specific Aims: Aim 1: To investigate the role of ApoE2 in neurons and astrocytes derived from isogenic hiPSCs. Aim 2: To define the role of ApoE2 in neurons and astrocytes derived through direct reprogramming and gene editing. Aim 3: To determine the relationship of ApoE2 genotype with gene expression levels in human brains. The proposed studies will likely help to define the neuroprotective roles of ApoE2 in the development of AD pathological features, and to uncover novel mechanisms underlying ApoE2 protective effects, which could lead to the development of novel ApoE2-based therapeutic strategies for AD.